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Influence of the addition of pollen and brewer's yeast on growth of

Saccharomyces cerevisiae in honey-must

Article  in  International Food Research Journal · January 2015

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Aniela Pinto Kempka Rosa Cristina Prestes Dornelles

State University of Santa Catarina, Brazil, Pinhalzinho Universidade Federal de Santa Maria
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 International Food Research Journal 22(3): 1288-1292 (2015)
Journal homepage: http://www.ifrj.upm.edu.my

Influence of the addition of pollen and brewer’s yeast on growth of

Saccharomyces cerevisiae in honey-must
*
Kempka, A.P., Frühauf, M., Pagliarini, M.A., Matiello, J.A., Fachinello, F. and
Prestes, R.C.

Universidade do Estado de Santa Catarina, Department of Food Engineering and Chemical

1

Engineering, BR 282 Km 573.7, s/n, 89.870-000, Pinhalzinho-SC, Brazil

2
Universidade Federal de Santa Maria, Department of Science and Food Technology, Avenida
Roraima, 1000, 97.105-900, Santa Maria-RS, Brazil

Article history Abstract

Received: 15 May 2014
Received in revised form:
23 September 2014
Accepted: 27 September 2014
Keywords
Honey
Supplement of nitrogen
Cellular growth
S. cerevisiae
Honey can be considered an excellent source of carbohydrates for fermentation processes, but
low concentrations of nitrogen are found in this substrate, having therefore, a possible need
for supplemental. This study aimed to verify the growth profiles of Saccharomyces cerevisiae
in honey-must supplemented with pollen or brewer’s yeast. Were made experiments with
concentrations 15-75 g/L of supplements and these were compared to an experiment without
addition of supplements. The elemental analysis showed that the amount of nitrogen present in
the brewer’s yeast is 2.85 times higher than the pollen present in the content. In relation to the
growth of S. cerevisiae it has been found that the lower concentrations tested led to higher growth
both for the pollen as for brewer’s yeast. The experiments added with brewer’s yeast reached
the maximum growth in 8 hours of fermentation and the experiments added pollen reached the
maximum growth in 12 hours of fermentation. The results showed that supplementation favors
the cell growth when compared to the experiment without supplementation.
© All Rights Reserved

Introduction may have different flavors depending on the floral

Honey, one of the oldest and most traditional
sweetening foods, has been reported to contain about
200 substances. This natural product is essentially
a concentrated aqueous solution of different
carbohydrates, including fructose, glucose, maltose,
sucrose, and other oligo- and polysaccharides
(Escuredo et al., 2013). The quality of honey is
determined by their sensory, physical and chemical
properties. Its physical and chemical properties
depend on the nectar and pollen floral source, color,
flavor, moisture and protein content and sugars
(Azeredo et al., 2003).
All honeys share certain general characteristics,
including a moisture content below 20%, a sugar
content of 70–80%, an ash content ranging from 0.1%
to 0.2%, and a pH between 3.8 and 4.7. Proteins,
free amino acids (principally proline), organic acids,
aromatics, and vitamins and minerals are minor
components and several enzymes are important
components of honey such as α-glucosidase,
β-glucosidase, amylase and glucose oxidase (Won et
al., 2008; Roldán et al., 2011).
Honey is a substance that has been used for
centuries to make beverages can be fermented to
produce different types of mead and spirits that
source of honey and additives and yeast used in
fermentation (Gupta & Sharma, 2009). For contain
low concentrations of protein and amino acids,
in fermentation processes may be necessary the
supplement these compounds.
For supplementation of protein content with the
purpose to produce fermented honey can be used as
pollen, which is collected from flowers as a source
of proteins, lipids, vitamins and minerals for the
survival of bees (Roldán et al., 2011). Commercial
brewer’s yeast is inactive yeast (dead yeast cells
with no leaving power) remaining after the brewing
process. It is an inexpensive nitrogen source with
good nutritional characteristics and a very bitter
taste, generally recognized as safe (GRAS) (Ferreira
et al., 2010).
The yeast (Saccharomyces cerevisiae) is used
by brewers repeatedly (usually 4-6 times) and is
the second main byproduct of the brewing industry
(brewer’s yeast). Its use is still limited, being basically
used as animal feed, receiving little attention as a
marketable commodity, and its disposal is often an
environmental problem. Various attempts have been
made to utilize them in biotechnological processes
such as, for example, the production of value-added
compounds such as ethanol, or as substrates for

*Corresponding author.
Email: aniela.kempka@udesc.br
Tel: +55 49 33663971
1289 Kempka et al./IFRJ 22(3): 1288-1292
the cultivation of micro-organisms, or simply raw
material for the extraction of compounds (Ferreira et
al., 2010). The objective of this study was to verify
the growth profiles of S. cerevisiae using different
concentrations of pollen and brewer’s yeast in the
honey-must and evaluate these possible nutritional
supplements as alternatives to the micro-organism.
Materials and Method
The experimental analyzes were performed at the
Bioprocess Laboratory and Microbiology Laboratory
in Department of Food Engineering - State
University of Santa Catarina. The Elemental Analysis
(Elementar CHNS) was performed by combustion
in the Laboratory of Soil and Sustainability of the
Department of Animal Science - State University of
Santa Catarina. The method is based on complete
combustion of a sample (1g) of known mass of
organic material, which contains mainly carbon (C),
hydrogen (H), nitrogen (N), sulfur (S) and oxygen
(O) and subsequent analysis of gas resulting from
the combustion process, essentially carbon dioxide
(CO2), water (H2O), nitrogen oxides (NOx) and sulfur
dioxide (SO2). For obtain the values of proteins, we
multiplied the value of the percentage of nitrogen by
6.25 (reference value).
Supplementation and determination of growth of
Saccharomyces cerevisiae
For verify the influence of pollen concentration
and the concentration of brewer’s yeast, the following
experiments were performed: control- C (without
pollen or brewer’s yeast), E1, E2, E3, E4 and E5
containing 15 g/L, 30 g/L, 45 g/L, 60 g/L and 75 g/L
of the pollen or brewer’s yeast , respectively, totaling
11 experiments. For obtaining of the fermentation
medium, was made the dilution of the honey, obtaining
a percentage of soluble solids of the 18°Brix. After,
has made the correct of the acidity to pH 3.6. The
concentrations of the pollen or brewer’s yeast were
added to each experiment and subsequently it was
inoculated with 15 g/hL of the S. cerevisiae. The
incubation occurred in an incubator at 25ºC for 12
hours (Roldan et al., 2011). Samples were withdrawn
each 2 hours to determine the growth of yeast.
The growth of S. cerevisiae was accomplished
using the methodology proposed by Mendes-Ferreira
et al. (2010) with modifications. Samples of the 10
mL were centrifuged for 10 minutes at 3,000 rpm in
pre-weighed tubes. Subsequently, the supernatant
was exhausted and dried in an incubator for 24 hours
at 100°C. The samples, after cooling, were weighted
and the mass of cells determined by the weight
difference. It was determined the mass of cells before
the start of fermentation to be discounted this value.
Determination of kinetic parameters
The kinetic parameters determined for the growth
of S. cerevisiae in pollen and in brewer’s yeast were:
the specific growth rate, which is the change of the
natural log of the cell number density with time and
the time required to double the population, called
the doubling time, shown in Equations 1 and 2,
respectively (Doran, 1995).
(1)
(2)
where X is the cell concentration (g/L); X0 is the initial
cell concentration (g/L), µ is the specific growth rate
(h-1), µm is the maximum specific growth rate (h-1), td
is the time (hours), td is the doubling time.
Statistical analysis
Data were analyzed using the Statistica 10.0®
(Statsoft Inc.). Significant differences (p<0.05)
between means were identified using Tukey
procedures. All the experiments were carried out in
duplicate.
Results and Discussion
It is found that in relation to the nitrogen content
(results of elemental analysis) the brewer’s yeast
corresponded to 46.56% of protein and the pollen
corresponded to 16.31% of protein. As for the
remaining of the elements, the values found were
presented near. According to Roldan et al. (2011),
the pollen contains, in addition to sugars, 7.4% of the
moisture, approximately 20% of the protein (close to
that found in the present study), 6% fat and 2.2% ash,
plus vitamins, minerals and carotenoids. The proline,
aspartic acid, phenylalanine and glutamic acid are
primary amino acid existing in the pollen. Cells
from yeast (brewer’s yeast) are used as a supplement
because it contains 45% to 60% of the protein. Yeast
cells also contain lipids, vitamins and minerals (Chae
et al. 2001).
In Figures 1 and 2 are shown the growth profiles
of S. cerevisiae in honey-must added of pollen and
brewer’s yeast, respectively. It is observed in Figure
1 that after 12 hours of fermentation, the experiment
containing 30 g/L of pollen presented the highest
growth of yeast, followed by the experiment with 15
g/L. The experiment showed the lowest growth in 12
hours was with the addition of 75 g/L of pollen. It is
also verified that the experiments with 45, 60 and 75
 Kempka et al./IFRJ 22(3): 1288-1292
Figure 1. Profiles of the growth of Saccharomyces
cerevisiae (g/L) in honey-must containing different
concentrations of pollen
Figure 2. Profiles of the growth of Saccharomyces
cerevisiae (g/L) in honey-must containing different
concentrations of brewer's yeast
g/L of pollen there was a decrease in the growth of
the 10th to the 12th hour.
In Figure 2, for growth in 12 hours, the experiment
that showed higher cell mass was the experiment with
30 g/L with brewer’s yeast followed by the C and the
experiment with addition of 15 g/L of the brewer’s
yeast. To verify the statistical difference between
results obtained using pollen and brewer’s yeast in 8,
10 and 12 hours, was held the Tukey Test, with 95%
reliability.
Table 1 shows the results for the experiments
with addition of pollen. It was found that between the
times of 8, 10 and 12 hours (for the same experiment),
there was statistical difference (p<0.05) between
the averages of the results of cell growth. For the
experiments C, with addition of 15 g/L of pollen and
with addition of 30 g/L of pollen, the highest value of
the cell growth was obtained for the 12-hour time, and
the values statistically different between themselves
(p<0.05). For the experiments with addition of 45
g/L, 60 g/L and 75 g/L of pollen, the maximum
values of cell growth were attained in 10 hours, also
statistically different between themselves (p<0.05).
The results of the experiments with addition of
1290
Table 1. Medium of growth of Saccharomyces
cerevisiae in honey-must (control- C) and the honey-
must added pollen to 8, 10 and 12 hours.
a
Means ± standard deviation followed by different lowercase
letters, in the column, are different at the 95% level of confidence
between hours. Means followed by different capital letters, in
the line, are different at the 95% level of confidence between
samples (for the same concentration of the pollen)
the brewer’s yeast are shown in Table 2. For times
of 8, 10 and 12 hours, we found (as compared to the
same experiment) that only statistically different
(p<0.05) between the mean values for cell growth
for the experiment with addition of the 45 g/L with
brewer’s yeast (maximum growth at 8 hours). For
other experiments, it can be affirmed that the time of
8 hours is sufficient in order to obtain the maximum
in cells of S. cerevisiae. Furthermore, comparing
the experiments at time for 8 hours, it is found that
maximum growth occurred for the experiment with
addition of 30 g/L of brewer’s yeast, with the highest
value obtained statistically different the other values,
followed the experiment with addition of 15 g/L of
brewer’s yeast.
Therefore, the experiments with addition of
pollen reached the maximum concentration of cells
in 12 hours and process for experiments with addition
of brewer’s yeast, the maximum concentration of
cells was obtained for the 8 hour time corresponding
to the experiment adding 30 g/L for both. According
Mendes-Ferreira et al. (2010), although more studies
are needed, the results of their study suggest a reduced
yeast fermentative ability, and thereby increased risk
of delayed or arrested fermentations, is due to factors
other than nitrogen limitation in honey-musts, the
same occurred in the present study when compared
the experiment C with the experiments supplemented.
It was found by elemental analysis that the amount
of nitrogen present in the brewer’s yeast is 2.85 times
higher than the amount of nitrogen present in the
1291 Kempka et al./IFRJ 22(3): 1288-1292
Table 2. Medium of growth of Saccharomyces cerevisiae
in honey-must (control-C) and the honey-must added
brewer's yeast to 8, 10 and 12 hours.
a
Means ± standard deviation followed by different lowercase
letters, in the column, are different at the 95% level of confidence
between hours. Means followed by different capital letters, in
the line, are different at the 95% level of confidence between
samples (for the same concentration of the brewer's yeast).
pollen. In relation to the growth of S. cerevisiae, the
lowest concentrations tested of the supplements (15
g/L and 30 g/L) led to higher growth, regardless of
supplementation with pollen or brewer’s yeast. The
experiments added with brewer’s yeast reached the
maximum growth at 8 hours of fermentation. For the
experiments added pollen, the maximum growth of
was reached at 12 hours.
Kempka and Mantovani (2013) in a study on
the production of mead observed a reduction in
96-hour of the fermentation with addition of 1%
of pollen compared to experiments without added,
demonstrating that nutrients, especially the protein
contained in pollen can increase the velocity the
fermentation process. Table 3 shows the kinetic
parameters of growth of S. cerevisiae in the
supplemented media. It is found that the values of the
specific growth rate for the experiments supplemented
with pollen were higher than the control experiment
(C) for concentrations of 15 g/L and 75 g/L. For the
experiments supplemented with brewer’s yeast, the
values of the specific growth rate for the experiments
supplemented with 15g/L, 30 g/L and 75 g/L were
higher than experiment C.
Regarding the doubling time, it is found in Table
3 that for the experiments with 15 g/L and 75 g/L
of pollen, the values of this parameter were lower
compared to the experiment C, the same occurring to
those supplemented with experiments 15 g/L, 30g/L
and 75 g/L of brewer’s yeast. It is also verified that
the experiment with 45 g/L of brewer’s yeast had the
Table 3. Maximum specific growth rates, doubling time
and yields for the experiments with addition of pollen and
brewer's yeast
highest doubling time (approximately 3 times greater
than the C), which may indicate an inhibitory effect
of this supplement on an intermediate concentration.
The lowest doubling time also occurred for the
experiment with the addition of brewers yeast (30
g/L), with doubling times 6 times lower than the time
shown by the experiment C and 2.5 times lower than
the experiment with 75 g/L (second lowest doubling
time).
For the two supplements, as there is an increased
concentration, is also an increase of the doubling time
and thereafter a decline, indicating that intermediate
concentrations (the concentrations tested) can lead
to the development of some inhibitory effect on S.
cerevisiae. In this way, it appears that both the pollen,
a product with higher added value, such as brewer’s
yeast, featured as a byproduct, can be used to produce
biomass of S. cerevisiae. Nevertheless, research is
still needed on the physiology and metabolism of
Saccharomyces cerevisiae under the particular harsh
honey-musts environment (Mendes-Ferreira et al.,
2010).
Conclusion
It was verified by elemental analysis that the
amount of nitrogen present in the brewer’s yeast
is 2.85 times higher than the pollen present in the
content. In relation to the growth of S. cerevisiae, the
lowest concentrations tested (15 and 30 g/L) have
led to higher growth, regardless of supplementation
with pollen or brewer’s yeast. The experiments added
with brewer’s yeast growth reached maximum at 8
hours of fermentation and for the experiments added
pollen, maximum growth was reached at 12 hours.
The pollen and brewer’s yeast can be considered
promising supplements for fermentation processes
 Kempka et al./IFRJ 22(3): 1288-1292 1292

where it requires nitrogen addition. The results of the

growth using brewer’s yeast as supplement presented
themselves in less time compared with pollen. This
product, often discarded or sent to animal feed, can
be used in fermentative processes, with satisfactory
results.

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